Pectin is a structural polysaccharide commonly found in the form of protopectin in plant cell walls. The backbone of pectin comprises α-1-4 linked galacturonic acid residues which are interrupted with a small number of 1,2 linked α-L-rhamnose units.
In addition, pectin comprises highly branched regions with an almost alternating rhamno-galacturonan chain. These highly branched regions also contain other sugar units (such as D-galactose. L-arabinose and xylose) attached by glycosidic linkages to the C3 or C4 atoms of the rhamnose units or the C2 or C3 atoms of the galacturonic acid units. The long chains of α-1-4 linked galacturonic acid residues are commonly referred to as “smooth” regions, whereas the highly branched regions are commonly referred to as the “hairy regions”.
Some of the carboxyl groups of the galacturonic residues are esterified (e.g. the carboxyl groups are methylated). Typically esterification of the carboxyl groups occurs after polymerisation of the galacturonic acid residues. However, it is extremely rare for all of the carboxyl groups to be esterified (e.g. methylated).
Usually, the degree of esterification will vary from 0–90%. If 50% or more of the carboxyl groups are esterified then the resultant pectin is referred to as a “high ester pectin” (“HE pectin” for short) or a “high methoxyl pectin”. If less than 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a “low ester pectin” (“LE pectin” for short) or a “low methoxyl pectin”. If 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a “medium ester pectin” (“ME pectin” for short) or a “medium methoxyl pectin”. If the pectin does not contain any—or only a few—esterified groups it is usually referred to as pectic acid.
The structure of the pectin, in particular the degree of esterification (e.g. methylation), dictates many of the resultant physical and/or chemical properties of the pectin. For example, pectin gelation depends on the chemical nature of the pectin, especially the degree of esterification. In addition, however, pectin gelation also depends on the soluble-solids content, the pH and calcium ion concentration. With respect to the latter, it is believed that the calcium ions form complexes with free carboxyl groups, particularly those on a LE pectin.
Pectic enzymes such as pectin methylesterases (EC 3.1.1.11), are classified according to their mode of attack on the galacturonan part of the pectin molecule. In more detail. PME activity produces free carboxyl groups and free methanol. The increase in free carboxyl groups can be easily monitored by automatic titration. In this regard, earlier studies have shown that some PMEs de-esterify pectins in a random manner, in the sense that they de-esterify any of the esterified (e.g. methylated) galacturonic acid residues on one or more than one of the pectin chains. Examples of PMEs that randomly de-esterify pectins may be obtained from fungal sources such as Aspergillus aculeatus (see WO 94/25575) and Aspergillus japonicus (Ishii et al 1980 J Food Sci 44 pp 611–14). Baron et al (1980 Lebensm. Wiss. M-Technol 13 pp 330–333) apparently have isolated a fungal PME from Aspergillus niger. This fungal PME is reported to have a molecular weight of 39000 D, an isoelectric point of 3.9, an optimum pH of 4.5 and a Km value (mg/ml) of 3.
In contrast, some PMEs are known to de-esterify pectins in a block-wise manner, in the sense that it is believed they attack pectins either at non-reducing ends or next to free carboxyl groups and then proceed along the pectin molecules by a single-chain mechanism, thereby creating blocks of un-esterified galacturonic acid units which can be calcium sensitive. Examples of such enzymes that block-wise enzymatically de-esterify pectin are plant PMEs. Up to 12 isoforms of PME have been suggested to exist in citrus (Pilnik W. and Voragen A. G. J. (Food Enzymology (Ed.: P. F. Fox); Elsevier; (1991); pp: 303–337). These isoforms have different properties.
Random or blockwise distribution of free carboxyl groups can be distinguished by high performance ion exchange chromatography (Schols et al Food Hydrocolloids 1989 6 pp 1115–121). These tests are often used to check for undesirable, residual PME activity in citrus juices after pasteurisation because residual PME can cause, what is called, “cloud loss” in orange juice in addition to a build up of methanol in the juice.
PME substrates, such as pectins obtained from natural plant sources, are generally in the form of a high ester pectin having a DE of about 70%. Sugar must be added to extracts containing these high ester PME substrates to provide sufficient soluble solids to induce gelling. Usually a minimum of 55% soluble solids is required. Syneresis tends to occur more frequently when the percentage soluble solids is less than 55%. When syneresis does occur, expensive additives must be used to induce gelling.
Versteeg et al (J Food Sci 45 (1980) pp 969–971) apparently have isolated a PME from orange. This plant PME is reported to occur in multiple isoforms of differing properties. Isoform I has a molecular weight of 36000 D, an isoelectric point of 10.0, an optimum pH of 7.6 and a Km value (mg/ml) of 0.083. Isoform II has a molecular weight of 36200 D, an isoelectric point of 11.0, an optimum pH of 8.8 and a Km value (mg/ml) of 0.0046. Isoform III (HMW-PE) has a molecular weight of 54000 D, an isoelectric point of 10.2, an optimum pH of 8 and a Km value (mg/ml) of 0.041. However, to date there has been very limited sequence data for such PMEs.
According to Pilnik and Voragen (ibid), PMEs may be found in a number of other higher plants, such as apple, apricot, avocado, banana, berries, lime, grapefruit, mandarin, cherries, currants, grapes, mango, papaya, passion fruit, peach, pear, plums, beans, carrots, cauliflower, cucumber, leek, onions, pea, potato, radish and tomato. However, likewise, to date there has been very limited sequence data for such PMEs.
A plant PME has been reported in WO-A-97/03574 (the contents of which are incorporated herein by reference). This PME has the following characteristics: a molecular weight of from about 36 kD to about 64 kD; a pH optimum of pH 7–8 when measured with 0.5% lime pectin in 0.15 M NaCl; a temperature optimum of at least 50° C.; a temperature stability in the range of from 10°—at least 40° C.; a Km value of 0.07%; an activity maximum at levels of about 0.25 M NaCl; an activity maximum at levels of about 0.2 M Na2SO4; and an activity maximum at levels of about 0.3 M NaNO3.
Another PME has been reported in WO 97/31102 (the contents of which are incorporated herein by reference).
PMEs have important uses in industry. For example, they can be used in or as sequestering agents for calcium ions. In this regard, and according to Pilnik and Voragen (ibid), cattle feed can be prepared by adding a slurry of calcium hydroxide to citrus peels after juice extraction. After the addition, the high pH and the calcium ions activate any native PME in the peel causing rapid de-esterification of the pectin and calcium pectate coagulation occurs. Bound liquid phase is released and is easily pressed out so that only a fraction of the original water content needs to be removed by expensive thermal drying. The press liquor is then used as animal feed.
As indicated above, a PME has been obtained from Aspergillus aculeatus (WO 94/25575). Apparently, this PME can be used to improve the firmness of a pectin-containing material, or to de-methylate pectin, or to increase the viscosity of a pectin-containing material.
It has also become common to use PME in the preparation of foodstuffs prepared from fruit or vegetable materials containing pectin—such as jams or preservatives. For example. WO-A-94/25575 further reports on the preparation of orange marmalade and tomato paste using PME obtained from Aspergillus aculeatus. 
JP-A-63/209553 discloses gels which are obtained by the action of PME, in the presence of a polyvalent metal ion, on a pectic polysaccharide containing as the main component a high-methoxyl poly α-1,4-D-galacturonide chain and a process for their production.
Pilnik and Voragen (ibid) list uses of endogenous PMEs which include their addition to fruit juices to reduce the viscosity of the juice if it contains too much pectin derived from the fruit, their addition as pectinase solutions to the gas bubbles in the albedo of citrus fruit that has been heated to a core temperature of 20° C. to 40° C. in order to facilitate removal of peel and other membrane from intact juice segments (U.S. Pat. No. 4,284,651), and their use in protecting and improving the texture and firmness of several processed fruits and vegetables such as apple (Wiley & Lee 1970 Food Technol 24 1168–70), canned tomatoes (Hsu et al 1965 J Food Sci 30 pp 583–588) and potatoes (Bartolome & Hoff 1972 J Agric Food Chem 20 pp 262–266).
Glahn and Rolin (1994 Food Ingredients Europe, Conf Proceedings pp 252–256) report on the hypothetical application of the industrial “GENU pectin type YM-100” for interacting with sour milk beverages. No details are presented at all on how GENU pectin type YM-100 is prepared.
EP-A-0664300 discloses a chemical fractionation method for preparing calcium sensitive pectin. This calcium sensitive pectin is said to be advantageous for the food industry.
The fruit ripening process has been extensively used as a model system to dissect genetically programmed organ differentiation. Studies with both non climacteric and climacteric fruits such as apples, bananas, tomatoes, pears, avocados and mangos, have provided evidence for differential gene expression during ripening. In particular, pectin degrading enzymes such as PME and polygalacturonase (PG) have been implicated in the biochemical conversion of pectic cell wall substances during fruit ripening. By way of example, enzymes, such as PME and PG, showing altered activities during ripening have been reported and the respective genes have been cloned (D. Grierson, 1985, CRC Critical Reviews in Plant Sciences 3, 113–132). The features of many of these genes have not been defined. However, one such gene has been shown to produce PG, the enzyme primarily responsible for degrading the cell wall. The synthesis of PG begins in tomatoes during early stages of ripening, and reaches a maximum at the soft red stage. This increase is paralleled closely by an increase in PG mRNA (Grierson et al. 1985, Plant 163, pp. 263–271). According to one hypothesis, PG solubilises pectic fragments during early ripening stages, and reduces the molecular weight of pectin fragments during later stages of ripening.